The global epidemic of chronic kidney disease (CKD) is progressing at an alarming rate. Up to 11% of the general population is affected in the US, Australia, Japan and Europe. There is a simultaneous steady increase of type II diabetes, and its associated kidney complications, particularly in India, China and South-East Asia, and kidney-related diseases are eluding present treatment options and resources. Histological and genetic data strongly implicate podocyte dysfunction in glomerular disease (Susztak and Bottinger 2006; Tryggvason et al. 2006).
One of the earliest events marking podocyte dysfunction is disruption of foot processes (FP) and slit diaphragms, which is thought to cause foot-process fusion and proteinuria (Susztak and Bottinger 2006). In most cases of CKD, the first clinical sign is proteinuria. If these early structural changes within podocytes are not reversed, progressive, severe damage occurs, leading to detachment of podocytes from the glomerular basement membrane (GBM). This results in scarring, obliteration of the urinary space, and development of segmental glomerulosclerosis and end stage renal failure. Rearrangement of the actin cytoskeleton which links the slit diaphragm, apical domain and sole plate, serves as a common denominator during foot process (FP) effacement. Thus, a better understanding of mechanisms controlling foot process formation in health and disease is essential to design better early diagnostics and therapies that intervene, while permanent damage may still be preventable.
Renal filtration occurs in the glomerulus, a specialized structure that ensures selectivity of the kidney filter so that water, electrolytes and waste products are passed into the urinary space, while essential plasma proteins are retained in the blood. A sign of glomerular dysfunction is the loss of protein in the urine termed proteinuria or nephrotic syndrome (defined as protein loss exceeding 3.5 grams/day). Proteinuria often leads to progressive renal failure, eventually requiring dialysis or kidney transplantation. Together with the GBM and the glomerular endothelial cells, podocytes form a key component of the kidney permeability barrier. Podocyte function depends on a complex cellular structure, which consists of a cell body, as well as major processes and foot processes (FP) as described above. The FPs of one podocyte are inter-digitated with those of its neighbors, and the intercellular space between adjacent foot processes is bridged by a slit diaphragm composed of the protein nephrin, which also represents the final barrier to protein loss. Thus, podocyte injury is tightly correlated with proteinuria.
Podocyte FPs contain an elaborate and dynamic actin-based cytoskeleton that is essential for their membrane morphogenesis and for establishing and maintaining the filtration barrier in the kidney (Faul et al. 2007). FPs contain a microfilament-based contractile apparatus composed of actin, myosin II, α-actinin, talin, and vinculin, which is linked to the GBM at focal contacts by an integrin α3β1 complex (Faul et al. 2007). The FP actin is organized in two principle forms: a podosome-like, cortical network of short branched actin filaments, and stress fibers composed of an actin:myosin core occupying the center of the FP (Ichimura et al. 2003). FP structure appears to be optimized for constant actin-driven morphological rearrangements, which are essential for glomerular filtration (Moeller and Holzman 2006).
Most forms of proteinuria and nephrotic syndrome involve a reduction of podocyte membrane extensions and transformation of podocyte FPs into a band of cytoplasm (i.e., FP effacement). Changes in FP morphology are primarily driven by reorganization of the actin cytoskeleton, which condenses into a thick bundle against the sole of podocyte foot processes. A number of proteins directly or indirectly alter podocyte cytoskeletal organization. For example, mutations in α-actinin-4, which cause a late-onset form of focal segmental glomerulosclerosis (FSGS), revealed the importance of structural actin binding proteins for podocyte function (Kaplan et al. 2000). Signals that originate at the slit diaphragm can directly influence the actin cytoskeleton in podocytes (Jones et al. 2006; Moeller et al. 2004).
It has been reported that cell focal adhesion turnover is mediated through dynamin-clathrin-dependent endocytosis of activated β1 integrins, and that knockdown of either dynamin II or both clathrin adaptors AP-2 and disabled-2 (DAB2) blocks β1 integrin internalization leading to impaired focal adhesion disassembly and cell migration (Chao and Kunz, 2009).
FP effacement during nephrotic syndrome is a migratory event (Reiser et al. 2004). Cultured podocytes contain all three major categories of actin structures required for cell migration: lamellipodia, filopodia and contractile actin stress fibers. Cultured podocytes also express all known differentiation markers characteristic of podocytes including: nephrin, podocin, CD2AP, synaptopodin, as well as known components of the slit diaphragm such as ZO-1, P-cadherin, α-, β-, and γ-catenin (Mundel et al. 1997; Saleem et al. 2002). Indeed, podocytes have been extensively used to study known actin binding and bundling proteins (e.g., α-actinin 4 and synaptopodin (Asanuma et al. 2005; Asanuma et al. 2006). The cortical actin web that underlies formation of lamellipodia and filopodia in cultured podocytes appears to be equivalent to the short-branched actin web in the vicinity of the plasma membrane observed by EM in podocytes in vivo (Ichimura et al. 2003). Similarly, actin-myosin stress fibers observed in cultured podocytes are likely to be equivalent to non-branched stress fibers occupying the center of FP in vivo (Ichimura et al. 2003).
Cytoskeletal dynamics are often controlled by the Rho family of small GTPases. At the leading edge of cells, Rac1 and Cdc42 promote cell motility through the formation of cortical actin, which in turn promotes motility through the formation of lamellipodia and filopodia, respectively. In contrast, RhoA promotes the formation of contractile actin-myosin-containing stress fibers in the cell body. RhoA signaling plays an important role in regulating the actin cytoskeleton in podocytes. Thus, synaptopodin, an actin-binding protein expressed in podocytes (Mundel et al. 1997) induces stress-fiber formation by extending the lifetime of active RhoA (Asanuma et al. 2006). The exact roles of Rac1 and Cdc42 signaling for podocyte structure and function are less well understood.
It has been reported that in some mouse models of nephrotic syndrome, preservation of dynamin is sufficient to counteract early stages of foot processes effacement and proteinuria (Sever et al. 2007). Dynamin is a large GTPase enzyme that severs membrane-bound clathrin-coated vesicles. The clathrin-mediated endocytic pathway is of special interest to biomedical researchers because it is involved in internalizing activated receptors, sequestering growth factors, antigen presentation, cytokinesis, synaptic transmission and as an entry route for a variety of pathogens. Dynamin comprises three major isoforms: dynamin I (neurons); dynamin II (ubiquitous) and dynamin III (neurons and testes) (Cousin and Robinson 2001). Common to all are five domains, a GTPase domain (required for vesicle fission), a middle domain (MD, of unknown function), pleckstrin homology domain (PH, targeting domain and potentially a GTPase inhibitory module), a GTPase effector domain (GED, which controls dynamin self-assembly into rings), and a proline-rich domain (PRD, which interacts with proteins containing an SH3 domain and is the main site for dynamin I and III phosphorylation in vivo).
Dynamin is best known for its roles in clathrin-mediated endocytosis at the plasma membrane and synaptic vesicle endocytosis in neurons (Sever et al. 2000b). A number of studies indicate that dynamin has additional roles, including regulation of the actin cytoskeleton through molecular mechanisms that are poorly understood (Schafer 2004). Dynamin's role in regulation of the actin cytoskeleton has been attributed to its interactions with known actin binding and regulatory proteins such as profilin, Nck and cortactin (Orth and McNiven 2003; Schafer 2004). A previous study has also indicated that dynamin is essential for formation of functional FPs in podocytes (Sever et al. 2007).
Dynamin exhibits unique biochemical characteristics distinct from other GTPases, such as high molecular weight (MW=100 kDa) and atypically low affinity for GTP (Km=˜10 μM). Dynamin exists in three main states—basal, ring or helix—and its GTPase activity increases stepwise upon transition to each state. More particularly:
a) In its “basal” state dynamin is in equilibrium between monomer, dimers and homotetramers (Muhlberg et al. 1997), and has a “basal” GTPase rate of ˜1 min−1.
b) Dynamin dimers or tetramers can further self-assemble into higher-order structures resembling “rings” that have an outer diameter of about 50 nm and an inner opening of about 30 nm (Hinshaw and Schmid, 1995). This typically occurs above 500 nM dynamin in vitro. The rings are not always closed and the diameter can vary between systems. Ring formation is promoted by dialysis of dynamin into low salt buffers and occurs with high concentrations of dynamin of around 0.5-1 micromolar. Ring formation stimulates dynamin's GTPase activity about 10 fold (Warnock et al. 1996). The increase in the rate of GTP hydrolysis is due to activation of intramolecular GTPase activating domain within dynamin that only becomes active when dynamin tetramers come together (Sever et al. 1999). A dynamin mutant has been reported that is predicted to live longer in the ring form—dynR725A is a mutant impaired in stimulated rate of GTP hydrolysis (Sever et al. 2000a).
c) In the presence of an assembly template dynamin can further assemble into a “helix” in vitro. The helix assembly templates include phospholipid liposomes, lipid nanotubes or microtubules. The helix appears to be an extension of the individual ring structure into a highly elongated helical structure, much like a spring. Helix formation stimulates dynamin's GTPase activity 100-1000 fold (Warnock et al. 1996). The stimulated rate of GTP hydrolysis in turn drives dynamin disassembly in vitro, and leads to loss of positive cooperativity for GTP-binding (Sever et al. 2006).
There is an emerging new field of dynamin pharmacology with the development of small-molecule inhibitors specific for the dynamin family of GTPases as powerful new tools with which to study cellular endocytosis in these systems. Small molecule dynamin inhibitors have attracted widespread attention and have been used to study endocytosis and other aspects of membrane dynamics in a variety of cellular systems (Macia et al. 2006). These inhibitors offer many distinct advantages over traditional means of dynamin inhibition in cells by expression of dynamin GTPase mutants or by small interfering RNA (siRNA)-mediated dynamin knockdown which cannot be used to study rapid cellular effects. Small molecule, cell-permeable inhibitors are able to rapidly block endocytosis in minutes and are readily reversible (Macia et al. 2006; Quan et al. 2007).
The first reported dynamin inhibitors were long chain ammonium salts such as myristyl trimethyl ammonium bromide (MiTMAB), octadecyltrimethyl ammonium bromide (OcTMAB) (Hill et al. 2004) and dimeric tyrphostins such as Bis-T (Hill et al. 2005). Later a series of room temperature ionic liquids (RTILs) (Zhang et al. 2008) and dynasore (Macia et al. 2006) were reported. Finally, indole-based inhibitors termed “dynoles” (Hill et al. 2009) and iminochromene-based inhibitors termed “iminodyns” have been reported (Hill et al. 2010). Most studies screening for dynamin inhibitors use GTPase assays whereby dynamin is maximally stimulated, and likely to be in its helical state. Some of the most potent inhibitors from each of these series are also potent and reversible inhibitors of endocytosis in cells (Quan et al. 2007; Hill et al. 2009; Hill et al 2010).